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Occurrence of flavonoids in Ophrys (Orchidaceae) flower parts
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Flora 203 (2008) 602–609 www.elsevier.de/flora
Occurrence of flavonoids in Ophrys (Orchidaceae) flower parts Anastasia Kariotia, Christine K. Kitsakib, Stella Zygourakib, Marouska Zioborab, Samah Djeddia, Helen Skaltsaa, Georgios Liakopoulosa,b, a
Department of Pharmacognosy and Chemistry of Natural Products, School of Pharmacy, Panepistimiopolis, Zografou, 15771, Athens, Greece b Laboratory of Plant Physiology, Department of Agricultural Biotechnology, Agricultural University of Athens, Iera Odos 75, Botanikos, 11855, Athens, Greece Received 25 April 2007; accepted 26 September 2007
Abstract The flower parts (i.e. sepals, labellum, gynostemium and isolated pollinia) of four Ophrys species (Ophrys argolica, Ophrys apifera, Ophrys cornuta, and Ophrys delfinensis [O. argolica O. cornuta]), native in Greece, were examined for the presence of polyphenolic compounds. The chemical composition was studied by chromatographic and spectroscopic techniques (UV–vis and 1D and 2D NMR), whereas the localization of the phenolic compounds was depicted by epifluorescence microscopy. The chemical composition was highly variable among the different flower parts. Pollinia and sepals contained the highest concentrations of flavonoids, while the labellum contained the lowest. Among species, O. apifera showed the highest (up to 6-fold) concentration of flavonoid compounds compared with the other three species. Microscopic observations showed that flavonoids are localized in the protoplast, especially in proximity of the nucleus. They are concentrated on cuticle and cell walls of epidermal cells and in parenchyma cells of the sepals. The pollinia contained high concentrations of flavonols which were deposited in the pollen grains. Three kaempferol (Km) glycosides, Km 3-O-b-D-glucoside, Km 3-O-b-D-rutinoside and Km 3-O-b-D-rhamnoside, were the dominant compounds in the pollinia. The above results suggest a UV-protective role for flavonoids in the flower parts of these plants. Additionally, the pollen-specific flavonols could also be related to the flavonol-depended male fertility known from other plant species. r 2008 Elsevier GmbH. All rights reserved. Keywords: Flavonoids; Flower; Ophrys sp.; Orchidaceae; Pollen fertility; Ultraviolet radiation
Introduction Phenolic compounds include plant secondary metabolites that determine to a large extent the characterCorresponding author at: Laboratory of Plant Physiology, Department of Agricultural Biotechnology, Agricultural University of Athens, Iera Odos 75, Botanikos, 11855, Athens, Greece. Tel.: +30 210 5294281; fax: +30 2105 294286. E-mail address: [email protected] (G. Liakopoulos).
0367-2530/$ - see front matter r 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.flora.2007.09.009
istics of the plant–environment interface (Harborne, 1993). The presence of phenolic compounds in epidermal tissue (Day, 1993; Hutzler et al., 1998; Manetas, 1999), cuticles (Karabourniotis and Liakopoulos, 2005; Kraus et al., 1997; Liakopoulos et al., 2001; Stavroulaki et al., 2007) and accessories (Karabourniotis et al., 1998; Skaltsa et al., 1994) is customary. Owing to their intense absorbance in the ultraviolet region and their high chemical stability (Rozema et al., 1997), surface or near surface depositions of phenolic compounds may act as
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an effective screen against harmful radiation (Caldwell et al., 1998). It is well documented that phenolic compounds contribute to both pre-existing and induced chemical defense of plant surfaces against biotic enemies (Seigler, 1998). Moreover, a number of studies have shown that in some plant species (both monocots and dicots) the presence of pollen flavonols is essential for the germination and proper growth of the pollen tube (Taylor and Jorgensen, 1992; Taylor and Hepler, 1997; Vogt and Taylor, 1995; Ylstra et al., 1994). In some species, pollen originating from mutants deficient in branch-point enzymes of the flavonoid biosynthetic pathway, show inability to germinate (Mo et al., 1992; Taylor and Jorgensen, 1992). Orchids are considered to be evolutionary advanced plant species and this is especially apparent in the reproduction process (Dressler, 1990). They demonstrate a complex flower construction and reproductive physiology and likely these characters are accompanied by an intricate phytochemistry. Orchid anthocyanins have drawn much attention (Strack et al., 1989), but reports on the chemistry of colourless flavonoids are limited. Williams (1979) surveyed orchids belonging to 75 genera for leaf flavonoid compounds. No reports exist for the localization and chemical composition of orchid flower flavonoids. In the present work, we studied the polyphenol chemistry of orchid flowers of four species belonging to the Ophrys genus. In particular, we focused on the tissue-specific localization of flavonoids in Ophrys flowers in order to identify their possible ecophysiological roles.
Materials and methods Plant material Natively grown plants of four Ophrys species (Ophrys argolica Fleischm., Ophrys apifera Hudson, Ophrys cornuta Stev. Ex M. Bieb., and Ophrys delfinensis O. Danesch & E. Danesch [O. argolica O. cornuta]) were collected during spring of 2003 and 2004 from Southern Attiki and Achaia (Greece) as described elsewhere (Kitsaki et al., 2004). Flowers were removed from plants and the flower parts studied were isolated using a scalpel. Samples consisted of sepals, labellum, gynostemium, and pollinia (Fig. 1). Three replicates were used per plant species and per flower part.
HPLC analysis and isolation of pollinia flavonoids For analytical HPLC, samples (ca. 200–1000 mg fresh weight) were placed in a porcelain mortar, grounded with liquid nitrogen to a fine powder and extracted at
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Fig. 1. (A) flower and (B–D) various flower parts of O. argolica. (B) labellum, (C) gynostemium, and (D) pollinia.
room temperature with 80% methanol (10 ml) using a pestle. The extract was centrifuged at 2600g for 15 min and the supernatant was concentrated in vacuum at 30 1C under nitrogen until it reached a 2–3 ml volume. The sample was then partitioned in acetonitrile: n-hexane 4:6 (v/v) to remove the non-polar constituents. The lower phase was concentrated in vacuum at 30 1C under nitrogen. Samples were stored at 30 1C until required for HPLC analyses. Aliquots were diluted to match the mobile phase and filtered through Chromafil RC-20/25 membrane filters (Macherey-Nagel, Du¨ren, Germany) prior to HPLC analysis. Analyses were performed in a Jasco HPLC system equipped with an LG-980-02 tertiary low-pressure gradient unit, a PU-980 pump and a UV-970 ultraviolet detector (Jasco Corporation, Tokyo, Japan). For analytical HPLC, samples were injected into an APEX ODS 5 mm, 25.0 4.6 mm2 analytical column in line with a 1 cm APEX ODS 5 mm guard column (Jones Chromatography Ltd., Mid Glamorgan, UK) using a 7725i injection valve (Reodyne, Rohnhert Park, CA USA) via a 20 ml sample loop. The analytical column was kept at 30 1C using a 7971 column heater (Jones Chromatography Ltd.). The mobile phase was degassed with helium. Solvent mixtures were, A: 5% aqueous sol. formic acid; B: acetonitrile; and C: methanol. The following linear gradient elution was used: 96:2:2 (A:B:C), initial conditions; isocratic for 5 min; gradient to 80:15:5 in 25 min; isocratic for 5 min; gradient to 79:16:5 in 5 min; isocratic for 5 min; gradient to 75:20:5 in 5 min; gradient
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to 40:20:40 in 5 min; gradient to 0:0:100 in 5 min; isocratic for 10 min. Re-establishment of initial conditions was done by transition to 96:2:2 in 5 min and equilibrating for 10 min. Mobile phase was delivered at 1.2 ml min 1; detection was performed at 300 nm. Chromatograms were captured in a PC system using Borwin Chromatographic Software, ver. 1.21.60 (JMBS Development, Fontaine, France). Flavonoids were identified with reference to pure standards and on-line UV-absorption spectra. For structural identification of the pollinia flavonoids, pollinia samples (1.0 g fresh weight) were extracted twice in 100% methanol and twice in 50% methanol. Both extracts were combined resulting in a residue of 0.43 g, which was chromatographed on Sephadex LH-20 column (Amersham Biosciences AB, Uppsala, Sweden) using methanol as eluent to yield 49 fractions of 10 ml, further combined to six groups (A–F). Fraction E (34.9 mg) was identified as kaempferol 3-O-b-D-glucoside. Fractions F (35.9 mg) was further purified by HPLC and afforded kaempferol 3-O-b-D-glucoside (22.0 mg), kaempferol 3-O-b-D-rutinoside (traces), and kaempferol 3-O-b-D-rhamnoside (traces). For preparative HPLC, fractions were chromatographed in a Kromasil C18 preparative column using isocratic elution (methanol:5% aqueous sol. acetic acid 40:60) at a flow rate 1.5 ml min 1. 1H and 2D NMR spectra were recorded in CD3OD, on Bruker DRX 400 instrument. Chemical shifts are given in ppm (d) and the spectra were referenced against undeuterated solvent. UV spectra were recorded on a Shimadzu UV-160A spectrophotometer, according to Mabry et al. (1970). For TLC, Merck silica gel 60 F254 (Art. 5554) and Merck cellulose (Art. 5552) plates were used. Detection was facilitated by UV-light and appropriate spray reagents [vanillinH2SO4 on silica gel; Neu’s reagent (diphenylboric acid 2aminoethyl ester; Sigma–Aldrich Co., St. Louis, MO, USA) on cellulose (Neu, 1957)].
Epifluorescence microscopy Intact stigmata and pollinia as well as fresh crosssections made by hand of sepals and labellum samples were prepared. All samples, except for pollinia, were treated with Neu’s reagent in 0.1% (w/v) ethanolic solution to induce fluorescence, incubated for 5 min, washed with distilled water and used directly for microscopic observations. Treatment with the above reagent allows the histochemical visualization of flavonoids via the secondary green-yellow fluorescence emitted upon excitation with blue light (Hutzler et al., 1998; Karabourniotis et al., 1998; Schnitzler et al., 1996). Pollinia samples were observed without fluorescence induction since their autofluorescence was particularly strong. Observations were made in a Zeiss
Axiolab microscope equipped with an HBO 50 UVlamp. Filter combinations (exciter filter/chromatic beam splitter/barrier filter) were G365/FT395/LP420 (UV 365 nm excitation) and BP450-490/FT510/LP520 (blue light excitation), (Carl Zeiss Jena GmbH, Germany). Microphotographs were taken using a Cybershot DSCS75 digital camera (SONY Corporation, Japan).
Results Distribution of flavonoids revealed by epifluorescent microscopy Labellum cross-sections were taken at the location where the type of pigmentation of the epidermal cells changes from non-pigmented (yellowish) to pigmented (cyanic) (Fig. 2A) creating the characteristic macroscopic patterns of the labellum (Fig. 1B). Observations under the epifluorescent microscope showed that nonpigmented epidermal cells emit strong fluorescence while pigmented cells do not emit any detectable fluorescence (Fig. 2B and C). This concerns for both yellow-green (Fig. 2B) and blue fluorescence (Fig. 2C), which, under the specific conditions, characterizes flavonoids and phenylpropanoid-related structures, respectively (Hutzler et al., 1998). On the other hand, both pigmented and non-pigmented cells exhibited strong fluorescence derived from the cuticle (Fig. 2B and C). Closer observations of pigmented cells under bright-field microscope revealed that the anthocyanic pigments are organized in dense aggregates, termed anthocyanic vacuolar inclusions (Markham et al., 2000), which occupy a significant portion of the cell volume (Fig. 2D). Examination of cross-sections of sepals showed that epidermal cells emit strong yellow fluorescence originating from the protoplast and especially the nucleus (Fig. 2F). Strong yellow fluorescence is also emitted from the cell walls and the cuticle of these cells under blue excitation light (Fig. 2G). The internal parenchyma cells of the sepals also fluoresce but the light emitted is relatively weaker compared with that of the epidermal cells. This observation includes both the protoplast as a whole and the nucleus (Fig. 2F). Pollinia viewed under epifluorescent microscope exhibited very strong green autofluorescence essentially derived from the pollen grains (Fig. 2E), indicating the presence of flavonoids (Urushibara et al., 1992). The results of the microscopic observations of samples from all four species were essentially the same (data not shown).
HPLC analyses We conducted chromatographic analyses of sepal, labellum, gynostemium together with pollinia extracts,
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Fig. 2. (A–C) Hand-cut cross-sections of O. argolica labellum at the adaxial epidermis viewed under bright field (A), or epifluorescent microscope with blue (B) or ultraviolet (C) excitation light. Microphotograph (A) depicts non-pigmented (left side) and anthocyanic (right side) epidermal cells. Note in (B) and (C) that yellow-green or blue fluorescence is emitted from nonpigmented cells only (white arrow) while no fluorescence is emitted from pigmented cells (black arrow). Cuticular fluorescence is emitted from both types of cells (white arrowheads). (D) Hand-cut cross-sections of O. argolica labellum at the adaxial epidermis viewed under bright field microscope showing anthocyanic epidermal cells containing anthocyanic vacuolar inclusions (arrows). (E) Intact pollinia of O. argolica under epifluorescent microscope emitting strong green autofluorescence upon excitation with blue light. (F, G) Hand-cut cross-sections of O. argolica sepal viewed under epifluorescent microscope with blue excitation light. In (F), note the intense yellow fluorescence emitted from the nuclei of the epidermal cells (arrows) and the relatively weaker emitted from the parenchyma cells (arrowheads). In (G), the fluorescence is emitted from both the cuticle (arrows) and the cell wall (arrowhead).
in order to study the chemical composition and the quantitative differences of phenolic substances among these structures. Fig. 3(A–C) depicts the HPLC traces of these extracts. Additionally, we have studied the chemical composition of the pollinia alone in order to assess their contribution to the overall chemical composition of the gynostemium (Fig. 3D). According to our results, the three flower parts show distinctly different polyphenol profiles. Sepals contained high concentration of flavonoids and the chemical profile was composed of numerous structures (Fig. 3A). The same conclusion was drawn for the gynostemium (Fig. 3C), but the labellum showed much lower concentrations and relatively fewer compounds (Fig. 3B). In the gynostemium, three dominant flavonoid compounds were observed in notably high concentrations (Fig. 3C). By analysing isolated pollinia, we found that these flavonoids are exclusively located in the pollinia being the dominant compounds in this flower part also (Fig. 3D). In order to elucidate the chemical structures of these flavonoids, we conducted preparative analyses which evidenced presence of three kaempferol glycosides, kaempferol 3-O-b-D-glucoside, kaempferol 3-O-bD-rutinoside and kaempferol 3-O-b-D-rhamnoside. Further co-chromatography of the isolated compounds
with pollinia extracts confirmed their identity in the pollinia extracts (Fig. 3D). According to the quantitative analyses (Fig. 4), sepals and gynostemium were the richest parts while the labellum was the poorest, concerning flavonoids. Among the four Ophrys species examined, O. apifera contained the higher concentration of flavonoids in both sepals and labellum while no difference was observed in the gynostemium (Fig. 4).
Discussion Polyphenol distribution in the main flower parts of the Ophrys species examined shows that a significant fraction of these compounds is located in surface (cuticle and cell wall of epidermal cells) or near-surface (epidermal protoplasts) cellular structures. Compared with other plant parts, labellum epidermal cells emitted weak fluorescence, especially in anthocyanic cells, as judged from the fluorescence micrographs. This may indicate that anthocyanic cells contain lesser amounts of colourless polyphenols or that the anthocyanic molecules mask the existence of colourless polyphenols by reducing the transmission of excitation light or/and
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Fig. 4. Concentration of flavonoids in the flower parts of the four Ophrys species. Bars are means of three observations7standard error of the mean.
Fig. 3. Analytical HPLC chromatograms of methanolic extracts of different flower parts of O. cornuta: (A) sepal, (B) labellum, (C) gynostemium (including pollinia) and (D) isolated pollinia. In (C), numbers denote the three flavonoids found in the pollinia; 1. kaempferol 3-O-b-D-glucoside, 2. kaempferol 3-O-b-D-rutinoside, 3. kaempferol 3-O-b-D-rhamnoside. Traces (A–C) are normalized per unit of fresh weight and are quantitatively comparable.
fluorescence. However, the results of the chemical analyses support the possibility that protoplasts of anthocyanic cells contain far less flavonoids and phenylpropanoids compared with non-pigmented cells. This result indicates that anthocyanins and colourless phenolics can substitute each other in UV-protection. It is probable, therefore, that UV-protection of the labellum is largely owed to the high concentration of epidermal anthocyanins. Although anthocyanins have a lower UV-absorbance compared with colourless polyphenols (Caldwell et al., 1983), their abundance as vacuolar inclusions (see Markham et al., 2000) in the
labellum epidermis renders colourless UV-absorbing phenolics as not so necessary. Moreover, the particular anatomy of the outer cell walls of the epidermal cells, which protrude to the exterior (Fig. 2A–D) most likely reflect and scatter a significant part of the incident radiation. Judged from the microscopic observations and the chemical analyses, sepals are well-resourced of phenolic constituents which appear aptly localized to attenuate the incident UV-radiation. Cuticular layers of the epidermal cells emitted strong yellow-green fluorescence which is indicative of the presence of flavonoids. The deposition of phenolic compounds, including flavonoids, in the cuticle of aerial plant parts is welldocumented (Harborne and Williams, 2000; Karabourniotis and Liakopoulos, 2005). Strong fluorescence was also emitted from the cell walls and the protoplasts of sepal epidermises. According to Markham et al. (2001), flavonoids in the epidermal cells of sepals from a number of species are distributed in all cellular compartments which include the cytoplasm, vacuole and cell walls. Liakopoulos et al. (2006) showed that cuticular waxes and cell walls of Olea europaea leaf hairs show flavonoid-related UV-absorbing capacity which is comparable to that of the soluble fraction. Also, in this study, strong yellow fluorescence was derived from the perinuclear region of epidermal cells. The association of polyphenols with the nucleus (Grandmaison and Ibrahim, 1996; Karabourniotis et al., 1998; Sheahan, 1996) indicates the need for protection of these sensitive targets in particular. A distinctly intense (auto)fluorescence was derived from the pollinia. According to Urushibara et al. (1992) and Agati et al. (2002), green fluorescence indicates the presence of flavonoids alone or in combination with phenylpropanoids. Careful observation indicated that the fluorescing compounds are deposited in the pollen
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grains since the grains were apparent from their stronger fluorescence emission compared with the waxy connective material of the pollinia. Flavonoids are universal constituents among the reproductive structures (i.e. spores, pollen, etc.) of virtually all terrestrial plant taxa (Rosema et al., 2001). Pollen flavonoids may have a protective role against UV radiation. To prevent damage from solar UV radiation, the pollen wall is an effective UV-screen. For example, Brassica pollen wall screens out more than 80% of the incident ultraviolet radiation (Demchick and Day, 1996). Despite the very strong and protective pollen wall, a considerable fraction of incident UV-B radiation, up to 20%, may be transmitted from the pollen wall into the protoplasm (Demchik and Day, 1996), thus causing damage to pollen DNA and other sensitive targets. Flavonoids located into the protoplast may offer additional protection to intact pollen. Moreover, flavonoids may protect the pollen during germination when the protoplast is no more surrounded by the pollen wall (Rosema et al., 2001). Between species, O. apifera had higher concentrations of flavonoids in the sepals and labellum, possibly indicating the necessity for stronger UV-B protection. The above could be attributed to the inhabitancy of this species to higher altitudes (up to 1800 m a.s.l.) compared with the other three species (Delforge, 1995). Early studies have shown the role of specific flavonoids in pollen grain germination (Mo et al., 1992; Sedgley, 1975; Ylstra et al., 1994). Untreated petunia pollens contain pollen-specific glycosylated flavonols (Pollak et al., 1993; Vogt and Taylor, 1995). These are originally synthesized in the tapetal cells of the anther and taken up as aglycons by the developing pollen grains where they are converted to the corresponding glycosides (Miller et al., 1999; Vogt and Taylor, 1995). These structures are believed to be directly involved in male fertility in this, and also other diverse plant species (Taylor and Hepler, 1997), perhaps acting as signal molecules during germination (Guyon et al., 2000). Pollak et al. (1993) formulated the hypothesis that upon pollen germination, 3-O-glycosylated derivatives of quercetin and kaempferol are converted back to their corresponding aglycones which are the final active forms that promote germination and pollen tube growth. Further evidence for this type of mechanism was published later (Miller et al., 1999; Xu et al., 1997). Biochemical complementation of flavonol-deficient Petunia pollen showed that the range of flavonoids that can restore germination is restricted to flavonols with unsubstituted hydroxyl groups at positions 3 and 7 of the flavonoid skeleton (Vogt et al., 1995). According to the same authors, exogenous supply of kaempferol resulted in full restoration of the deficient pollen. In our study, three kaempferol-3-O-glycosides were identified as the dominant polyphenols of the pollinia. Analytical
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HPLC chromatograms confirmed the presence of the isolated kaempferol glycosides in the pollinia of all four species examined. The presence of quercetin and kaempferol derivatives in leaves of the Ophrys genus have been reported previously (Williams, 1979). It may be suggested that in the Ophrys species examined, the above structures may, in the context of molecular structure, be related to male fertility. To verify if pollen flavonoids are indeed a prerequisite for pollen germination and successful pollen tube growth in the Ophrys genus, the examination of mutants deficient in branchpoint enzymes of the flavonol biosynthetic pathway is necessary. To our knowledge, such mutants are not yet available.
Acknowledgements This study is dedicated to the memory of Mr. Yiannis Th. Kalopissis, an agriculturist recognized for its contribution to the knowledge of Greek orchid flora. Authors wish to thank Assistant Professor Theophanis Constantinidis (Laboratory of Systematic Botany, Agricultural University of Athens, Greece) for identification of plant material.
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Occurrence of flavonoids in Ophrys (Orchidaceae) flower parts Anastasia Kariotia, Christine K. Kitsakib, Stella Zygourakib, Marouska Zioborab, Samah Djeddia, Helen Skaltsaa, Georgios Liakopoulosa,b, a
Department of Pharmacognosy and Chemistry of Natural Products, School of Pharmacy, Panepistimiopolis, Zografou, 15771, Athens, Greece b Laboratory of Plant Physiology, Department of Agricultural Biotechnology, Agricultural University of Athens, Iera Odos 75, Botanikos, 11855, Athens, Greece Received 25 April 2007; accepted 26 September 2007
Abstract The flower parts (i.e. sepals, labellum, gynostemium and isolated pollinia) of four Ophrys species (Ophrys argolica, Ophrys apifera, Ophrys cornuta, and Ophrys delfinensis [O. argolica O. cornuta]), native in Greece, were examined for the presence of polyphenolic compounds. The chemical composition was studied by chromatographic and spectroscopic techniques (UV–vis and 1D and 2D NMR), whereas the localization of the phenolic compounds was depicted by epifluorescence microscopy. The chemical composition was highly variable among the different flower parts. Pollinia and sepals contained the highest concentrations of flavonoids, while the labellum contained the lowest. Among species, O. apifera showed the highest (up to 6-fold) concentration of flavonoid compounds compared with the other three species. Microscopic observations showed that flavonoids are localized in the protoplast, especially in proximity of the nucleus. They are concentrated on cuticle and cell walls of epidermal cells and in parenchyma cells of the sepals. The pollinia contained high concentrations of flavonols which were deposited in the pollen grains. Three kaempferol (Km) glycosides, Km 3-O-b-D-glucoside, Km 3-O-b-D-rutinoside and Km 3-O-b-D-rhamnoside, were the dominant compounds in the pollinia. The above results suggest a UV-protective role for flavonoids in the flower parts of these plants. Additionally, the pollen-specific flavonols could also be related to the flavonol-depended male fertility known from other plant species. r 2008 Elsevier GmbH. All rights reserved. Keywords: Flavonoids; Flower; Ophrys sp.; Orchidaceae; Pollen fertility; Ultraviolet radiation
Introduction Phenolic compounds include plant secondary metabolites that determine to a large extent the characterCorresponding author at: Laboratory of Plant Physiology, Department of Agricultural Biotechnology, Agricultural University of Athens, Iera Odos 75, Botanikos, 11855, Athens, Greece. Tel.: +30 210 5294281; fax: +30 2105 294286. E-mail address: [email protected] (G. Liakopoulos).
0367-2530/$ - see front matter r 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.flora.2007.09.009
istics of the plant–environment interface (Harborne, 1993). The presence of phenolic compounds in epidermal tissue (Day, 1993; Hutzler et al., 1998; Manetas, 1999), cuticles (Karabourniotis and Liakopoulos, 2005; Kraus et al., 1997; Liakopoulos et al., 2001; Stavroulaki et al., 2007) and accessories (Karabourniotis et al., 1998; Skaltsa et al., 1994) is customary. Owing to their intense absorbance in the ultraviolet region and their high chemical stability (Rozema et al., 1997), surface or near surface depositions of phenolic compounds may act as
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an effective screen against harmful radiation (Caldwell et al., 1998). It is well documented that phenolic compounds contribute to both pre-existing and induced chemical defense of plant surfaces against biotic enemies (Seigler, 1998). Moreover, a number of studies have shown that in some plant species (both monocots and dicots) the presence of pollen flavonols is essential for the germination and proper growth of the pollen tube (Taylor and Jorgensen, 1992; Taylor and Hepler, 1997; Vogt and Taylor, 1995; Ylstra et al., 1994). In some species, pollen originating from mutants deficient in branch-point enzymes of the flavonoid biosynthetic pathway, show inability to germinate (Mo et al., 1992; Taylor and Jorgensen, 1992). Orchids are considered to be evolutionary advanced plant species and this is especially apparent in the reproduction process (Dressler, 1990). They demonstrate a complex flower construction and reproductive physiology and likely these characters are accompanied by an intricate phytochemistry. Orchid anthocyanins have drawn much attention (Strack et al., 1989), but reports on the chemistry of colourless flavonoids are limited. Williams (1979) surveyed orchids belonging to 75 genera for leaf flavonoid compounds. No reports exist for the localization and chemical composition of orchid flower flavonoids. In the present work, we studied the polyphenol chemistry of orchid flowers of four species belonging to the Ophrys genus. In particular, we focused on the tissue-specific localization of flavonoids in Ophrys flowers in order to identify their possible ecophysiological roles.
Materials and methods Plant material Natively grown plants of four Ophrys species (Ophrys argolica Fleischm., Ophrys apifera Hudson, Ophrys cornuta Stev. Ex M. Bieb., and Ophrys delfinensis O. Danesch & E. Danesch [O. argolica O. cornuta]) were collected during spring of 2003 and 2004 from Southern Attiki and Achaia (Greece) as described elsewhere (Kitsaki et al., 2004). Flowers were removed from plants and the flower parts studied were isolated using a scalpel. Samples consisted of sepals, labellum, gynostemium, and pollinia (Fig. 1). Three replicates were used per plant species and per flower part.
HPLC analysis and isolation of pollinia flavonoids For analytical HPLC, samples (ca. 200–1000 mg fresh weight) were placed in a porcelain mortar, grounded with liquid nitrogen to a fine powder and extracted at
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Fig. 1. (A) flower and (B–D) various flower parts of O. argolica. (B) labellum, (C) gynostemium, and (D) pollinia.
room temperature with 80% methanol (10 ml) using a pestle. The extract was centrifuged at 2600g for 15 min and the supernatant was concentrated in vacuum at 30 1C under nitrogen until it reached a 2–3 ml volume. The sample was then partitioned in acetonitrile: n-hexane 4:6 (v/v) to remove the non-polar constituents. The lower phase was concentrated in vacuum at 30 1C under nitrogen. Samples were stored at 30 1C until required for HPLC analyses. Aliquots were diluted to match the mobile phase and filtered through Chromafil RC-20/25 membrane filters (Macherey-Nagel, Du¨ren, Germany) prior to HPLC analysis. Analyses were performed in a Jasco HPLC system equipped with an LG-980-02 tertiary low-pressure gradient unit, a PU-980 pump and a UV-970 ultraviolet detector (Jasco Corporation, Tokyo, Japan). For analytical HPLC, samples were injected into an APEX ODS 5 mm, 25.0 4.6 mm2 analytical column in line with a 1 cm APEX ODS 5 mm guard column (Jones Chromatography Ltd., Mid Glamorgan, UK) using a 7725i injection valve (Reodyne, Rohnhert Park, CA USA) via a 20 ml sample loop. The analytical column was kept at 30 1C using a 7971 column heater (Jones Chromatography Ltd.). The mobile phase was degassed with helium. Solvent mixtures were, A: 5% aqueous sol. formic acid; B: acetonitrile; and C: methanol. The following linear gradient elution was used: 96:2:2 (A:B:C), initial conditions; isocratic for 5 min; gradient to 80:15:5 in 25 min; isocratic for 5 min; gradient to 79:16:5 in 5 min; isocratic for 5 min; gradient to 75:20:5 in 5 min; gradient
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to 40:20:40 in 5 min; gradient to 0:0:100 in 5 min; isocratic for 10 min. Re-establishment of initial conditions was done by transition to 96:2:2 in 5 min and equilibrating for 10 min. Mobile phase was delivered at 1.2 ml min 1; detection was performed at 300 nm. Chromatograms were captured in a PC system using Borwin Chromatographic Software, ver. 1.21.60 (JMBS Development, Fontaine, France). Flavonoids were identified with reference to pure standards and on-line UV-absorption spectra. For structural identification of the pollinia flavonoids, pollinia samples (1.0 g fresh weight) were extracted twice in 100% methanol and twice in 50% methanol. Both extracts were combined resulting in a residue of 0.43 g, which was chromatographed on Sephadex LH-20 column (Amersham Biosciences AB, Uppsala, Sweden) using methanol as eluent to yield 49 fractions of 10 ml, further combined to six groups (A–F). Fraction E (34.9 mg) was identified as kaempferol 3-O-b-D-glucoside. Fractions F (35.9 mg) was further purified by HPLC and afforded kaempferol 3-O-b-D-glucoside (22.0 mg), kaempferol 3-O-b-D-rutinoside (traces), and kaempferol 3-O-b-D-rhamnoside (traces). For preparative HPLC, fractions were chromatographed in a Kromasil C18 preparative column using isocratic elution (methanol:5% aqueous sol. acetic acid 40:60) at a flow rate 1.5 ml min 1. 1H and 2D NMR spectra were recorded in CD3OD, on Bruker DRX 400 instrument. Chemical shifts are given in ppm (d) and the spectra were referenced against undeuterated solvent. UV spectra were recorded on a Shimadzu UV-160A spectrophotometer, according to Mabry et al. (1970). For TLC, Merck silica gel 60 F254 (Art. 5554) and Merck cellulose (Art. 5552) plates were used. Detection was facilitated by UV-light and appropriate spray reagents [vanillinH2SO4 on silica gel; Neu’s reagent (diphenylboric acid 2aminoethyl ester; Sigma–Aldrich Co., St. Louis, MO, USA) on cellulose (Neu, 1957)].
Epifluorescence microscopy Intact stigmata and pollinia as well as fresh crosssections made by hand of sepals and labellum samples were prepared. All samples, except for pollinia, were treated with Neu’s reagent in 0.1% (w/v) ethanolic solution to induce fluorescence, incubated for 5 min, washed with distilled water and used directly for microscopic observations. Treatment with the above reagent allows the histochemical visualization of flavonoids via the secondary green-yellow fluorescence emitted upon excitation with blue light (Hutzler et al., 1998; Karabourniotis et al., 1998; Schnitzler et al., 1996). Pollinia samples were observed without fluorescence induction since their autofluorescence was particularly strong. Observations were made in a Zeiss
Axiolab microscope equipped with an HBO 50 UVlamp. Filter combinations (exciter filter/chromatic beam splitter/barrier filter) were G365/FT395/LP420 (UV 365 nm excitation) and BP450-490/FT510/LP520 (blue light excitation), (Carl Zeiss Jena GmbH, Germany). Microphotographs were taken using a Cybershot DSCS75 digital camera (SONY Corporation, Japan).
Results Distribution of flavonoids revealed by epifluorescent microscopy Labellum cross-sections were taken at the location where the type of pigmentation of the epidermal cells changes from non-pigmented (yellowish) to pigmented (cyanic) (Fig. 2A) creating the characteristic macroscopic patterns of the labellum (Fig. 1B). Observations under the epifluorescent microscope showed that nonpigmented epidermal cells emit strong fluorescence while pigmented cells do not emit any detectable fluorescence (Fig. 2B and C). This concerns for both yellow-green (Fig. 2B) and blue fluorescence (Fig. 2C), which, under the specific conditions, characterizes flavonoids and phenylpropanoid-related structures, respectively (Hutzler et al., 1998). On the other hand, both pigmented and non-pigmented cells exhibited strong fluorescence derived from the cuticle (Fig. 2B and C). Closer observations of pigmented cells under bright-field microscope revealed that the anthocyanic pigments are organized in dense aggregates, termed anthocyanic vacuolar inclusions (Markham et al., 2000), which occupy a significant portion of the cell volume (Fig. 2D). Examination of cross-sections of sepals showed that epidermal cells emit strong yellow fluorescence originating from the protoplast and especially the nucleus (Fig. 2F). Strong yellow fluorescence is also emitted from the cell walls and the cuticle of these cells under blue excitation light (Fig. 2G). The internal parenchyma cells of the sepals also fluoresce but the light emitted is relatively weaker compared with that of the epidermal cells. This observation includes both the protoplast as a whole and the nucleus (Fig. 2F). Pollinia viewed under epifluorescent microscope exhibited very strong green autofluorescence essentially derived from the pollen grains (Fig. 2E), indicating the presence of flavonoids (Urushibara et al., 1992). The results of the microscopic observations of samples from all four species were essentially the same (data not shown).
HPLC analyses We conducted chromatographic analyses of sepal, labellum, gynostemium together with pollinia extracts,
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Fig. 2. (A–C) Hand-cut cross-sections of O. argolica labellum at the adaxial epidermis viewed under bright field (A), or epifluorescent microscope with blue (B) or ultraviolet (C) excitation light. Microphotograph (A) depicts non-pigmented (left side) and anthocyanic (right side) epidermal cells. Note in (B) and (C) that yellow-green or blue fluorescence is emitted from nonpigmented cells only (white arrow) while no fluorescence is emitted from pigmented cells (black arrow). Cuticular fluorescence is emitted from both types of cells (white arrowheads). (D) Hand-cut cross-sections of O. argolica labellum at the adaxial epidermis viewed under bright field microscope showing anthocyanic epidermal cells containing anthocyanic vacuolar inclusions (arrows). (E) Intact pollinia of O. argolica under epifluorescent microscope emitting strong green autofluorescence upon excitation with blue light. (F, G) Hand-cut cross-sections of O. argolica sepal viewed under epifluorescent microscope with blue excitation light. In (F), note the intense yellow fluorescence emitted from the nuclei of the epidermal cells (arrows) and the relatively weaker emitted from the parenchyma cells (arrowheads). In (G), the fluorescence is emitted from both the cuticle (arrows) and the cell wall (arrowhead).
in order to study the chemical composition and the quantitative differences of phenolic substances among these structures. Fig. 3(A–C) depicts the HPLC traces of these extracts. Additionally, we have studied the chemical composition of the pollinia alone in order to assess their contribution to the overall chemical composition of the gynostemium (Fig. 3D). According to our results, the three flower parts show distinctly different polyphenol profiles. Sepals contained high concentration of flavonoids and the chemical profile was composed of numerous structures (Fig. 3A). The same conclusion was drawn for the gynostemium (Fig. 3C), but the labellum showed much lower concentrations and relatively fewer compounds (Fig. 3B). In the gynostemium, three dominant flavonoid compounds were observed in notably high concentrations (Fig. 3C). By analysing isolated pollinia, we found that these flavonoids are exclusively located in the pollinia being the dominant compounds in this flower part also (Fig. 3D). In order to elucidate the chemical structures of these flavonoids, we conducted preparative analyses which evidenced presence of three kaempferol glycosides, kaempferol 3-O-b-D-glucoside, kaempferol 3-O-bD-rutinoside and kaempferol 3-O-b-D-rhamnoside. Further co-chromatography of the isolated compounds
with pollinia extracts confirmed their identity in the pollinia extracts (Fig. 3D). According to the quantitative analyses (Fig. 4), sepals and gynostemium were the richest parts while the labellum was the poorest, concerning flavonoids. Among the four Ophrys species examined, O. apifera contained the higher concentration of flavonoids in both sepals and labellum while no difference was observed in the gynostemium (Fig. 4).
Discussion Polyphenol distribution in the main flower parts of the Ophrys species examined shows that a significant fraction of these compounds is located in surface (cuticle and cell wall of epidermal cells) or near-surface (epidermal protoplasts) cellular structures. Compared with other plant parts, labellum epidermal cells emitted weak fluorescence, especially in anthocyanic cells, as judged from the fluorescence micrographs. This may indicate that anthocyanic cells contain lesser amounts of colourless polyphenols or that the anthocyanic molecules mask the existence of colourless polyphenols by reducing the transmission of excitation light or/and
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Fig. 4. Concentration of flavonoids in the flower parts of the four Ophrys species. Bars are means of three observations7standard error of the mean.
Fig. 3. Analytical HPLC chromatograms of methanolic extracts of different flower parts of O. cornuta: (A) sepal, (B) labellum, (C) gynostemium (including pollinia) and (D) isolated pollinia. In (C), numbers denote the three flavonoids found in the pollinia; 1. kaempferol 3-O-b-D-glucoside, 2. kaempferol 3-O-b-D-rutinoside, 3. kaempferol 3-O-b-D-rhamnoside. Traces (A–C) are normalized per unit of fresh weight and are quantitatively comparable.
fluorescence. However, the results of the chemical analyses support the possibility that protoplasts of anthocyanic cells contain far less flavonoids and phenylpropanoids compared with non-pigmented cells. This result indicates that anthocyanins and colourless phenolics can substitute each other in UV-protection. It is probable, therefore, that UV-protection of the labellum is largely owed to the high concentration of epidermal anthocyanins. Although anthocyanins have a lower UV-absorbance compared with colourless polyphenols (Caldwell et al., 1983), their abundance as vacuolar inclusions (see Markham et al., 2000) in the
labellum epidermis renders colourless UV-absorbing phenolics as not so necessary. Moreover, the particular anatomy of the outer cell walls of the epidermal cells, which protrude to the exterior (Fig. 2A–D) most likely reflect and scatter a significant part of the incident radiation. Judged from the microscopic observations and the chemical analyses, sepals are well-resourced of phenolic constituents which appear aptly localized to attenuate the incident UV-radiation. Cuticular layers of the epidermal cells emitted strong yellow-green fluorescence which is indicative of the presence of flavonoids. The deposition of phenolic compounds, including flavonoids, in the cuticle of aerial plant parts is welldocumented (Harborne and Williams, 2000; Karabourniotis and Liakopoulos, 2005). Strong fluorescence was also emitted from the cell walls and the protoplasts of sepal epidermises. According to Markham et al. (2001), flavonoids in the epidermal cells of sepals from a number of species are distributed in all cellular compartments which include the cytoplasm, vacuole and cell walls. Liakopoulos et al. (2006) showed that cuticular waxes and cell walls of Olea europaea leaf hairs show flavonoid-related UV-absorbing capacity which is comparable to that of the soluble fraction. Also, in this study, strong yellow fluorescence was derived from the perinuclear region of epidermal cells. The association of polyphenols with the nucleus (Grandmaison and Ibrahim, 1996; Karabourniotis et al., 1998; Sheahan, 1996) indicates the need for protection of these sensitive targets in particular. A distinctly intense (auto)fluorescence was derived from the pollinia. According to Urushibara et al. (1992) and Agati et al. (2002), green fluorescence indicates the presence of flavonoids alone or in combination with phenylpropanoids. Careful observation indicated that the fluorescing compounds are deposited in the pollen
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grains since the grains were apparent from their stronger fluorescence emission compared with the waxy connective material of the pollinia. Flavonoids are universal constituents among the reproductive structures (i.e. spores, pollen, etc.) of virtually all terrestrial plant taxa (Rosema et al., 2001). Pollen flavonoids may have a protective role against UV radiation. To prevent damage from solar UV radiation, the pollen wall is an effective UV-screen. For example, Brassica pollen wall screens out more than 80% of the incident ultraviolet radiation (Demchick and Day, 1996). Despite the very strong and protective pollen wall, a considerable fraction of incident UV-B radiation, up to 20%, may be transmitted from the pollen wall into the protoplasm (Demchik and Day, 1996), thus causing damage to pollen DNA and other sensitive targets. Flavonoids located into the protoplast may offer additional protection to intact pollen. Moreover, flavonoids may protect the pollen during germination when the protoplast is no more surrounded by the pollen wall (Rosema et al., 2001). Between species, O. apifera had higher concentrations of flavonoids in the sepals and labellum, possibly indicating the necessity for stronger UV-B protection. The above could be attributed to the inhabitancy of this species to higher altitudes (up to 1800 m a.s.l.) compared with the other three species (Delforge, 1995). Early studies have shown the role of specific flavonoids in pollen grain germination (Mo et al., 1992; Sedgley, 1975; Ylstra et al., 1994). Untreated petunia pollens contain pollen-specific glycosylated flavonols (Pollak et al., 1993; Vogt and Taylor, 1995). These are originally synthesized in the tapetal cells of the anther and taken up as aglycons by the developing pollen grains where they are converted to the corresponding glycosides (Miller et al., 1999; Vogt and Taylor, 1995). These structures are believed to be directly involved in male fertility in this, and also other diverse plant species (Taylor and Hepler, 1997), perhaps acting as signal molecules during germination (Guyon et al., 2000). Pollak et al. (1993) formulated the hypothesis that upon pollen germination, 3-O-glycosylated derivatives of quercetin and kaempferol are converted back to their corresponding aglycones which are the final active forms that promote germination and pollen tube growth. Further evidence for this type of mechanism was published later (Miller et al., 1999; Xu et al., 1997). Biochemical complementation of flavonol-deficient Petunia pollen showed that the range of flavonoids that can restore germination is restricted to flavonols with unsubstituted hydroxyl groups at positions 3 and 7 of the flavonoid skeleton (Vogt et al., 1995). According to the same authors, exogenous supply of kaempferol resulted in full restoration of the deficient pollen. In our study, three kaempferol-3-O-glycosides were identified as the dominant polyphenols of the pollinia. Analytical
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HPLC chromatograms confirmed the presence of the isolated kaempferol glycosides in the pollinia of all four species examined. The presence of quercetin and kaempferol derivatives in leaves of the Ophrys genus have been reported previously (Williams, 1979). It may be suggested that in the Ophrys species examined, the above structures may, in the context of molecular structure, be related to male fertility. To verify if pollen flavonoids are indeed a prerequisite for pollen germination and successful pollen tube growth in the Ophrys genus, the examination of mutants deficient in branchpoint enzymes of the flavonol biosynthetic pathway is necessary. To our knowledge, such mutants are not yet available.
Acknowledgements This study is dedicated to the memory of Mr. Yiannis Th. Kalopissis, an agriculturist recognized for its contribution to the knowledge of Greek orchid flora. Authors wish to thank Assistant Professor Theophanis Constantinidis (Laboratory of Systematic Botany, Agricultural University of Athens, Greece) for identification of plant material.
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